Kinases are one of the main classes of drug targets with at least 30 distinct targets now present in clinical trials4,5. The vast majority of these drugs, typically inhibitors of kinase activity, are being investigated for the treatment of cancer. Traditionally, kinase inhibitors were designed to inhibit ATP binding and thus prevent protein activity; most kinase drugs, known as type I inhibitors, mimic and bind to the ATP binding site, directly competing with ATP. Type I inhibitors stabilize the activation loop, an important structural element that determines the protein's activity, in an active conformation (FIG. 2, right). In contrast, type II inhibitors (e.g., Gleevec and sorafenib), which were first discovered serendipitously about a decade ago, cause the activation loop to shift to an inactive conformation (FIG. 2, left). They bind partly to the ATP binding site and partly to an additional hydrophobic pocket that is revealed in the inactive conformation.6-10 The intense current interest in type II inhibitors is the result of five main factors.4,5,11 First, the striking clinical success of Gleevec (imatinib), a type II inhibitor, in treating chronic-phase Chronic Myeloid Leukemia (CML) is fueling their demand. Second, the interactions of type II inhibitors in the hydrophobic pocket are more unique structurally, across kinases in the kinome, than those in the ATP binding pocket. As a result, type II inhibitors are expected to exhibit significantly better selectivity and slower off-rates in general. Third, type II inhibitors offer a route to expand the chemical space of kinase drugs because the scaffolds of many compound libraries have already been exploited for type I inhibitors. Fourth, there is a critical need for new type II inhibitors that can overcome mutational resistance; cocktails will likely become the dominant treatment paradigm. Fifth, although type II inhibitors have been identified against a handful of kinases, they have not been identified yet for the other ˜500 kinases, leaving open the tantalizing possibility of developing Gleevec-like drugs for many other cancers and diseases.
Unfortunately, although demand for novel type II inhibitors is intense, identifying them is difficult.4,12,13 They are often overlooked in traditional enzymatic assays because of their low affinity for active, phosphorylated kinases. The most direct way currently to identify type II inhibitors is by X-ray crystallography, whose throughput is, at best, about 100 co-structures per month. However, the throughput demand for a screen that can identify them is much higher. Therefore, a critical need exists for a new technique that is both high-throughput and can readily identify type II inhibitors.
The invention described herein addresses these problems and provides additional benefits as well.
Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles) are referenced. The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.